Field of the Invention
The present disclosure generally relates to control the length of overhangs present in double stranded DNAs.
Description of the Related Art
The process of joining the two ends of DNA strands is called ligation. DNA ligation is an essential step for cloning genes and DNA fragments or performing DNA recombinant technologies. One of the main factors that affect the efficiency of ligation is the type of the ends DNA strands have: blunt ends or cohesive ends with either 5′ or 3′ overhangs. Among them, the blunt and short cohesive ends tend to have a low efficiency.
The ligation may be performed either via a ligase dependent way in which the enzyme called ligase is used, or a ligase independent way. The latter is increasingly being used due to its short reaction time and high efficiency compared to the former.
US Patent Publication 2012-0283144 relates to a method to improve the efficiency of ligation and discloses the use of straight or branched diols or polyols with a molecular weight of 1000 Da or less in addition to ligases.
Examples of ligase independent methods include in vivo recombination based cloning and exonuclease based cloning. The latter is disclosed in U.S. Pat. No. 7,575,860. However, the methods employing exonucleases have a drawback that the ligation efficiency is heavily dependent on the overhang length. Thus there exist needs to develop ways to control the length of overhangs particularly for the application in the ligase independent cloning.